Amino acid sequence of a crystalline seed albumin (winged bean albumin-1) from Psophocarpus tetragonolobus (L.) DC. Sequence similarity with Kunitz-type seed inhibitors and 7S storage globulins

The complete amino acid sequence of winged bean albumin-1 (WBA-1) of Psophocarpus tetragonolobus (L.) DC has been determined. The protein consists of a single polypeptide chain of 175 amino acid residues, with one disulfide bond, corresponding to a molecular mass of 19333 Da. WBA-1 was found to be homologous with the Kunitz-type seed trypsin inhibitors. The similarity between WBA-1 and the trypsin inhibitors from soybean and winged bean was 38% and 28%, respectively; similarity was most marked in the C-terminal third of the sequence with identities of 47% and 37%, respectively. Significant similarity was found also between the 2S Kunitz-type proteins and the carboxy-terminal region of the 7S storage globulins, suggesting that these two groups of proteins are related and may have evolved from a common ancestral precursor. Circular dichroism measurements suggest a high content of beta sheet (52%) while secondary structure predictions based on amino acid sequence indicate a similar content and distribution of beta sheet to that found for soybean trypsin inhibitor by X-ray diffraction studies.     

7S globulins from Phaseolus vulgaris L.: Impact of structural aspects on the nutritional quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS-PAGE showed major bands corresponding to the phaseolin subunits (43-53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 degrees C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of beta-sheet and beta-turn structures, together with a contribution at 1687 cm(-1) that was assigned to intramolecular beta-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Formation of Pseudo- and Hybrid-11S Globulins from Subunits of Soybean and Broad Bean 11S Globulins

Pseudo- and hybrid-11S globulins were reconstituted from native acidic and basic subunits of soybean and broad bean 11S globulins. The subunit structures of these two globulins are known to be similar to each other. Pseudo-11S globulins were formed in combinations between glycinin acidic subunit (G-AS_{1 + 2}) and glycinin basic subunit (G-BS) and between legumin acidic subunit (L-ASII) and legumin basic subunit (L-BS). Hybrid-11S globulins were formed in combinations between G-AS_{1 + 2} and L-BS and between L-ASII and G-BS. The yields of the reconstituted 11S components of G-AS_{1 +2} + G-BS and G-AS_{1 + 2} + L-BS were lower than those of L-ASII + G-BS and L-ASII + L-BS. These pseudo- and hybrid-11S globulins were similar to native 11S globulins; they all consisted of reconstituted intermediary subunits which were composed of acidic and basic subunits linked by disufide bridges and had molecular weights similar to those of native 11S globulins. However, the dissociation-association behaviors of pseudo-glycinin and hybrid-11S globulins were different from those of native 11S globulins.     

7S Globulins from Phaseolus vulgaris L.: Impact of Structural Aspects on the Nutritional Quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS–PAGE showed major bands corresponding to the phaseolin subunits (43–53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 °C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of β-sheet and β-turn structures, together with a contribution at 1687 cm^?1 that was assigned to intramolecular β-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.     

Globulins enhance in vitro iron but not zinc dialysability: a study on six legume species.

The study was addressed to evaluate the in vitro iron and zinc dialysability from the globulin fraction of six legumes. Five legume species including white bean, mottled bean (Taylor bean), chickpea, lentil, lupin, and a modified mottled bean variety, selected by back-crossing to obtain seeds with globulins composed by G1 fraction only, were used. Globulins (G1 + G2) were extracted from the seeds and analysed for their in vitro iron and zinc dialysability. The highest globulin concentration was detected in lentil (89%). The percentage of globulins in the modified variety of Taylor bean (G1 only) was higher than that of the commercial variety (G1 + G2). The highest concentration of iron was found in Taylor bean globulins. The modified variety of Taylor bean contained 2.6-fold higher iron concentration than the whole seed, and the commercial variety had 1.8-fold higher iron only. The highest zinc concentration was found in lentil globulins. Also iron dialysability from globulins was markedly higher than that of the respective whole seed. The highest value of iron dialysability was found in lentil (10.8%). Zinc dialysability was generally high (above 20%), but no significant differences between whole seed and globulins were detected. The results showed that globulins enhanced iron but not zinc dialysability. Lupin and the modified variety of Taylor bean showed a different behaviour in terms of mineral dialysability compared to the other legumes. The amino acid composition of the digestion products of whole seeds and globulins failed to evidence any direct influence on iron and zinc availability.     

Effects of Nitrate Level on Nitrogen Metabolism in Winged Bean and Soya Bean

The effects of high (15 mM) and low (0.75 mM) solution nitratelevels on nitrogen metabolism in three genotypes (IL 7A, IL13 and IL 21) of winged beans [Psophocarpus tetragonolobus (L.)DC.] and one genotype (Williams) of soya bean [Glycine max (L.)Merrill] were investigated. Plants were grown for 42 days ina greenhouse in solution culture prior to sampling. The 15 mM nitrate treatment resulted in greater growth of allplant parts except roots. Growth of soya beans was more responsiveto nitrate level than was growth of winged beans. The high nitratelevel inhibited nodulation in all plants. The IL 13 and IL 21winged bean genotypes had similar nitrogenase activity (acetylenereduction per plant) as the soya bean and IL 7A winged beangenotype had lower activity. However, the IL 13 winged beangenotype had higher nitrogenase activity (acetylene reductionper unit nodule mass) than the other three genotypes which allhad similar activity. The 15 mM solution nitrate level stimulatedleaf and root nitrate reductase (NR) activity for all plants.All winged bean genotypes had higher leaf NR activity and higherpercentage reduced- and nitrate-nitrogen contents of leavesand stems compared with soya beans. However, total protein (reducednitrogen) was greater in soya beans when sampled indicatingthat more nitrate had been metabolized by soya beans than bywinged beans during the 42-day growth period. Psophocarpus tetragonolobus (L.) DC., winged bean, Glycine max (L.) Merrill, Soya bean, nitrate reductase, nitrogen fixation, nitrogenase activity, nodulation     

Generality and Diversity of Winged Bean (Psophocarpus tetragonolobus) Protein in Eight Various Lines

Protein components from eight lines of winged bean (Psophocarpus tetragonolobus) seeds which were originally introduced from Papua New Guinea, Indonesia, Nigeria, and Ishigaki, and cultivated in Okinawa and Fukuoka, were investigated. Two major peaks which had sedimentation coefficients, s₂₀, of about 2.5S and about 6.5S (6.0 to 6.6 for the 8 lines), and no larger component were observed in all specimens with more than 90% extraction. Electrophoretic profiles of the “6.5S” component(s) which was separated with Sepharose 6B column chromatography showed a main broad band and a few minor bands which seemed to be essentially similar among the eight lines of winged bean. Thus the “6.5S” protein surely could be regarded as the common storage protein in winged bean seeds. The subunit structure of the “6.5S” component(s) in SDS solution consisted of four major bands. The “2.5S” components were mixtures and combinations of various proteins which were distinctly different from one selection to another.     

早熟翼豆“833”选育初报

本文报道了早熟翼豆“833”品种的选育过程和主要性状。试验表明,翼豆“833”具有早熟,蛋白质含量高和固氮能力强等优点。在高纬度,夏季长日照的条件下能开花。豆荚的成熟期也相应提早。     

Studies of the infection of the winged bean by Synchytrium psophocarpi in Papua New Guinea

Infection of leaves and stems of Psophocarpus tetragonolobus by Synchytrium psophocarpi only occurred following spray inoculation of motile zoospore suspensions and incubation for a minimum of 12 h in polyethylene bags or a mist chamber. The incubation period was 7 days and generation time 22 days at temperatures of 31 ^oC max, 24 ^oC min and r.h. of 90% max, 70% min. Young, 1–2 day-old leaves were most susceptible; there was no infection on 10 day-old leaves and susceptibility was not increased by the removal of leaf waxes. No infection occurred when plants were grown from seed from infected pods, seed inoculated with zoospores or sporangia and seed sown in soil containing infected leaf debris. Resting spores were not found in infected tissues stored for 12 wk or in plant debris. S. psophocarpi did not infect Arachis hypogaea, Glycine max, Phaseolus aureus, P. coccineus, P. vulgaris, Pisum sativum, Psophocarpus scandens, Vicia faba, Vigna sesquipedalis and V. unguiculata. S. minutum did not infect winged bean. Inoculation confirmed the susceptibility of the winged bean lines UPS 62, UPS 122, UPS 126 and resistance of two Thai winged bean lines 1602/1 and 1611/2.     

Electron microscopy of seed-storage globulins

The quaternary structures of a range of seed globulins, including examples of both the so-called 7 S and 11 S types, have been examined by electron microscopy. The legume 7 S proteins, phaseolin (bean), beta-conglycinin (soybean), and vicilin (pea), appear as flat discs of diameter ca. 8.5 nm and thickness ca. 3.5 nm formed by association of three subunit domains. Phaseolin converts to an 18 S tetramer at acid pH, and images recorded under these conditions suggest that four of the 7 S protomer discs associate to form the faces of a regular tetrahedron. The classical 11 S seed globulins, cucurbitin (pumpkin) and legumin (pea), are approximately spherical molecules of diameter ca. 8.8 nm composed of six subunits. In contrast, the hexameric 10 S storage protein from lupin seed, conglutin gamma, appears toroidal in shape with outer diameter ca. 10.3 nm and thickness ca. 2.2 nm. These results indicate that constraints imposed on seed proteins by their role in sustaining the germinating plant may have allowed a variety of different globulin structures to accumulate in the protein-storage bodies of seeds.     

The Ricinus communis 2S albumin precursor: a single preproprotein may be processed into two different heterodimeric storage proteins

Summary TheRicinus communis (castor bean) 2S albumin is a heterodimer of glutamine-rich, disulphidelinked 4 and 7 kDa polypeptides. A cDNA library was constructed using mRNA from maturing castor bean endosperm as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present in the 2 S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor included two further glutamine-rich sequences which, in terms of their size and conserved cysteine residues typically found in seed proteins of the 2S albumin superfamily, possibly represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme is proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. The generation of a second heterodimer seems likely since polypeptide candidates for its small and large subunits were found in theRicinus 2S albumin fraction, and N-terminal protein sequencing confirmed the existence of the putative small subunit.     

Characterization of the acidic lectins from winged bean seed (Psophocarpus tetragonolobus(L.)DC)

The seeds of winged bean, Psophocarpus tetragonolobus(L.)DC, contain two distinct groups of lectins characterized by different erythrocyte hemagglutinating specificities and isoelectric points. Three acidic lectins (I, II, and III) (pI approximately 5.5) were purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP-Sephadex C-25. These lectins are glycoproteins with relative molecular mass of 54,000. The total carbohydrate content of the acidic lectins was 7% and was comprised of mannose, N-acetylglucosamine, fucose, and xylose in amounts corresponding to 9.2, 4.8, 1.6, and 7.0 mol/54,000 g, respectively. Electrophoresis in dodecyl sulfate, in the presence and absence of 2-mercaptoethanol, gave a single subunit of apparent relative molecular mass 30-32,000, somewhat higher than expected from the native relative molecular mass. On isoelectric focusing in 8 M urea the subunits of the acidic lectins did not show any significant charge heterogeneity as found for the winged bean basic lectins. The acidic lectins have very similar amino acid compositions. They contain essentially no half-cystine, 1-2 methionine residues, and are rich in acidic and hydroxy amino acids. The amino-terminal sequences of lectins II and III were identical while the amino-terminal sequence of lectin I contained five differences in the first 25 residues; the acidic lectins showed extensive sequence homology with the winged bean basic lectins, the other one-chain subunit lectins and the beta subunit of the two-chain subunit legume lectins. The acidic lectins agglutinated trypsinized human (type A, B, AB, and O) erythrocytes but not trypsinized rabbit erythrocytes. They were inhibited by various D-galactose derivatives and D-galactose-containing disaccharides and trisaccharides. N-Acetylgalactosamine was the best inhibitor, and the specificity appears to be directed to beta-D-galactosides. However, compared with winged bean basic lectins and soybean lectin, the winged bean acidic lectins show a low affinity for the inhibitory sugars.     

Dry bean protein functionality

Dry beans are an important source of proteins, carbohydrates, dietary fiber, and certain minerals and vitamins in the human food supply. Among dry beans, Phaseolus beans are cultivated and consumed in the greatest quantity on a worldwide basis. Typically, most dry beans contain 15 to 25% protein on a dry weight basis (dwb). Water-soluble albumins and salt-soluble globulins, respectively, account for up to 10 to 30% and 45 to 70% of the total proteins (dwb). Dry bean albumins are typically composed of several different proteins, including lectins and enzyme inhibitors. A single 7S globulin dominates dry bean salt soluble fraction (globulins) and may account for up to 50 to 55% of the total proteins in the dry beans (dwb). Most dry bean proteins are deficient in sulfur amino acids, methionine, and cysteine, and therefore are of lower nutritional quality when compared with the animal proteins. Despite this limitation, dry beans make a significant contribution to the human dietary protein intake. In bean-based foods, dry bean proteins also serve additional functions that may include surface activity, hydration, and hydration-related properties, structure, and certain organoleptic properties. This article is intended to provide an overview of dry bean protein functionality with emphases on nutritional quality and hydration-related properties.     

A predominantly hydrophobic recognition of H-antigenic sugars by winged bean acidic lectin: a thermodynamic study

The thermodynamics of binding of winged bean (Psophocarpus tetragonolobus) acidic agglutinin to the H-antigenic oligosaccharide (Fucalpha1-2Galbeta1-4GlcNAc-oMe) and its deoxy and methoxy congeners were determined by isothermal titration calorimetry. We report a relatively hydrophobically driven binding of winged bean acidic agglutinin to the congeners of the above sugar. This conclusion is arrived, from the binding parameters of the fucosyl congeners, the nature of the enthalpy-entropy compensation plots and the temperature dependence of binding enthalpies of some of the congeners. Thus, the binding site of winged bean acidic agglutinin must be quite extended to accommodate the trisaccharide, with non-polar loci that recognize the fucosyl moiety of the H-antigenic determinant.     

Crystallization and preliminary X-ray studies of psophocarpin B1, a chymotrypsin inhibitor from winged bean seeds

Psophocarpin B1 is a 20,000 Mr protein of winged bean (Psophocarpus tetragonolobus) seeds having chymotrypsin inhibitory activity. Single crystals of this protein suitable for X-ray crystallographic studies have been obtained by the vapour diffusion method using ammonium sulphate. The crystals are hexagonal, space group P6(4)22 or P6(2)22, cell dimensions a = b = 61 A, c = 210 A. They are stable to irradiation with X-rays and diffract to at least 2.6 A resolution.     

Microbial Degradation of α-Picolinic Acid, 2-Pyridinecarboxylic Acid

The biological effects of raw winged bean seeds were investigated with feeding experiments on rats, and the effects of lectin (phytohemagglutinin) present in the seeds are discussed. Administration of a 30% raw winged bean diet caused strong growth depression in young rats, and led to death within 10 ~ 20 days, inducing severe damage to the small intestine of the rats. Significant morphological changes of the intestinal mucosa were observed with a microscopic investigation. As the lethal effect was eliminated by autoclaving but not removed with supplementation of 0.5% l-methionine to the raw winged bean diet, the lectin was assumed to be closely related to the deleterious effects of raw winged bean. In vitro and in vivo digestion tests of the lectin revealed that the winged bean lectin had resistance to peptic, pancreatic and membrane digestions. The hemagglutinating activity was also detected in the intestinal mucosa and faeces from rats ingesting the raw winged bean or its purified lectin. The binding action of lection to mucosal epitheliums of the gastrointestinal tract is suggested to be the initial step of the deleterious effects induced by the winged bean lectin.     

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.     

Dry Bean Protein Functionality

ABSTRACT:?

Dry beans are an important source of proteins, carbohydrates, dietary fiber, and certain minerals and vitamins in the human food supply. Among dry beans, Phaseolus beans are cultivated and consumed in the greatest quantity on a worldwide basis. Typically, most dry beans contain 15 to 25% protein on a dry weight basis (dwb). Water-soluble albumins and salt-soluble globulins, respectively, account for up to 10 to 30% and 45 to 70% of the total proteins (dwb). Dry bean albumins are typically composed of several different proteins, including lectins and enzyme inhibitors. A single 7S globulin dominates dry bean salt soluble fraction (globulins) and may account for up to 50 to 55% of the total proteins in the dry beans (dwb). Most dry bean proteins are deficient in sulfur amino acids, methionine, and cysteine, and therefore are of lower nutritional quality when compared with the animal proteins. Despite this limitation, dry beans make a significant contribution to the human dietary protein intake. In bean-based foods, dry bean proteins also serve additional functions that may include surface activity, hydration, and hydration-related properties, structure, and certain organoleptic properties. This article is intended to provide an overview of dry bean protein functionality with emphases on nutritional quality and hydration-related properties.     

Cross-inoculation specificity ofPsophocarpus tetragonolobus (L.) DC

Summary Only legumes of the cowpea cross-inoculation group, including the winged bean (Psophocarpus tetragonolobus) were found to form nodules in a temperate zone soil with no previous history of legume cropping. Isolates from root nodules from uninoculated winged beans grown in the field only nodulated legumes in the cowpea cross-inoculation group.Rhizobium japonicum formed ineffective nodules with the winged bean. Contribution No.5852, Scientific Article No.A2802 of the Maryland Agricultural Experiment Station, Department of Botany.